Treatment of multiple sclerosis and neuromyelitis optica

ABSTRACT

The present disclosure provides for the diagnosis and prediction of neuromyelitis optica (NMO) in subject. It also provides for treatment of multiple sclerosis (MS) in a subject. Thus, in accordance with the present disclosure, there is provided a method for treating a subject having neuromyelitis optica (NMO) comprising administering to said subject an inhibitor of B-cell activating factor (BAFF) and/or an inhibitor or proliferating inducing ligand (APRIL).

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/020,756, filed Jul. 3, 2014, the entire contentsof which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates to fields of pathology, immunology andmolecular biology. More particularly, the present invention relates totreatment of certain forms of multiple sclerosis (MS) and neuromyelitisoptica (NMO).

2. Description of Related Art

In autoimmune diseases, the function of B-cells is highly nuanced. Thishas been illuminated by results from clinical trials of therapies thattarget B-cells in multiple sclerosis (MS) and systemic lupuserythematosus (SLE). Rituximab (anti-CD20), an antibody which killsB-cells, effectively reduces MS relapses. Yet, blockade of BAFF andAPRIL, two cytokines critical for B-cell survival and development, withAtacicept worsened disease activity in MS patients. This unexpectedresult of Atacicept in MS is in contrast to the success of blocking BAFFin SLE, where anti-BAFF therapy reduced disease flares.

These confounding results from the Rituximab, Atacicept, and anti-BAFFtrials in MS and SLE provide strong evidence that B-cells can have bothpro-inflammatory and anti-inflammatory functions depending on thecontext of the autoimmune disease. In fact, there are now severalstudies in mice and humans that have identified regulatory B-cellsubsets (Bregs) that have the capability to inhibit inflammation andautoimmunity. This paradoxical role of B-cells/BAFF/APRIL in MS comparedto other autoimmune diseases is also mirrored by the function ofβ-interferon (IFN). In SLE and neuromyelitis optica (NMO), IFN-βexacerbates disease flares. Yet in MS, recombinant IFN-β is a widelyused therapy and reduces disease activity. Strikingly, IFN-β increasesBAFF and APRIL expression in MS, lupus and NMO providing more evidencefor the paradoxical function of B-cells in these autoimmune diseases.

The inventor has developed two mouse models of experimental autoimmuneencephalomyelitis (EAE), the animal model for MS, which differ inresponse to IFN-β mimicking aspects of MS and NMO. He has published thatEAE induced with myelin specific T Helper 1 cells (TH1-EAE) has reduceddisease activity when treated with IFN-β and therefore and thereforemodels IFN-β responsive MS. On the other hand, EAE induced withmyelin-specific T Helper 17 cells (TH17-EAE) worsens when treated withIFN-β, modeling NMO and IFN-β non-responsive MS. The significance ofthis distinction is still not fully understood.

SUMMARY OF THE INVENTION

Thus, in accordance with the present disclosure, there is provided amethod for treating a subject having neuromyelitis optica (NMO)comprising administering to said subject an inhibitor of B-cellactivating factor (BAFF) and/or an inhibitor or proliferating inducingligand (APRIL). The administering may comprise intravenous,intra-arterial, subcutaneous, topical or oral administration. Theinhibitor or inhibitors may be administered more than once, such aschronically. The method may further comprise administering to saidsubject a second NMO therapy, such as a corticosteroid, an elastaseinhibitor, a gro-alpha inhibitor, azathioprine plus prednisone,mycophenolate mofetil plus prednisone, Rituximab, Mitoxantrone,intravenous immunoglobulin (IVIG), and Cyclophosphamide. The subject maysuffer from vision impairment, muscle impairment or both, and thesubject, following treatment, may exhibit an improvement in visionimpairment, muscle impairment or both. The subject may be a non-humanmammal or a human.

In another embodiment, there is provided a method for treating a subjecthaving interferon-resistant multiple sclerosis (MS) comprisingadministering to said subject an inhibitor of B-cell activating factor(BAFF) and/or an inhibitor or proliferating inducing ligand (APRIL). Theadministering may comprise intravenous, intra-arterial, subcutaneous,topical or oral administration. The inhibitor or inhibitors may beadministered more than once, such as chronically. The method may furthercomprise administering to said subject a second MS therapy, such as acorticosteroid, an elastase inhibitor, a gro-alpha inhibitor,azathioprine plus prednisone, mycophenolate mofetil plus prednisone,Rituximab, Mitoxantrone, intravenous immunoglobulin (IVIG), andCyclophosphamide. The subject may suffer from vision impairment, muscleimpairment or both, and the subject, following treatment, may exhibit animprovement in vision impairment, muscle impairment or both. The subjectmay be a non-human mammal or a human.

In still another embodiment, there is provided a method of identifying asubject having multiple sclerosis (MS) that will be resistant tointerferon therapy comprising (a) obtaining a sample from said subject;and (b) assessing levels of a TH17/granulocyte factor, a type 1interferon, B-cell activating factor (BAFF) and/or proliferatinginducing ligand (APRIL) in said sample, wherein a subject havingelevated TH17/granulocyte factors, type 1 interferons, BAFF and/or APRILlevels will not respond to interferon therapy. The method may furthercomprise administering to a subject having elevated levels an inhibitorof B-cell activating factor (BAFF) and/or an inhibitor or proliferatinginducing ligand (APRIL). The method may also further comprise providinga written communication of said level or levels.

The administering may comprise intravenous, intra-arterial,subcutaneous, topical or oral administration. The inhibitor orinhibitors may be administered more than once, such as chronically. Themethod may further comprise administering to said subject a second MStherapy, such as a corticosteroid, an elastase inhibitor, a gro-alphainhibitor, azathioprine plus prednisone, mycophenolate mofetil plusprednisone, Rituximab, Mitoxantrone, intravenous immunoglobulin (WIG),and Cyclophosphamide. The subject may suffer from vision impairment,muscle impairment or both, and the subject, following treatment, mayexhibit an improvement in vision impairment, muscle impairment or both.The subject may be a non-human mammal or a human.

Assessing may comprise an immunoassay, mass spectrometry or RT-PCR. TheTH17/granulocyte factors may be selected from the group consisting ofIL-17A, IL-17F, IL-8, Gro-alpha and/or CXCL5. The type 1 interferons mayinclude interferon-α and/or interferon-β. A TH17/granulocyte factorlevel may be elevated, a type 1 interferon level may be elevated, BAFFlevels may be elevated, APRIL levels may be elevated, or more than oneor all of a TH17/granulocyte factor, a type 1 interferon, BAFF and APRILlevel are elevated.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawing forms part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-D. Characterization of TH1 and TH17-EAE. (FIG. 1A) Diseasecourse of mice with TH1- and TH17-induced EAE. Disease was induced bythe transfer of myelin-specific TH1 or TH17 cells into healthyrecipients and paralysis was monitored daily. (FIG. 1B) Quantitative PCRreveals a significant increase in mRNA expression of BAFF and APRIL inspinal cords of TH17-EAE compared to TH1-EAE. (FIG. 1C) FACS analysis ofCNS infiltrating CD19+ B-cells and CD4+ T-cells in TH1- and TH17-inducedEAE. (FIG. 1D) Significant increase in frequency of B-cells but not CD4+T-cells in TH17-EAE compared to TH1-EAE.

FIGS. 2A-C. Effects of TACI-Ig on TH17-EAE. (FIG. 2A) Mice with TH17-EAEwere treated with TACI-Ig (100 μg/dose) or PBS beginning at the peak ofdisease. Arrows depict dosing. (FIG. 2B) FACS analysis of peripheralblood B-cells in EAE mice treated with TACI-Ig or PBS compared tohealthy mice. TACI-Ig treatment restores the percentage of immatureIgM^(hi)/IgD^(lo) B-cells. (FIG. 2C) TACI-Ig significantly increasesimmature B-cell percentage in TH17-EAE. P-values were determined bystudent's T-test.

FIGS. 3A-B. Serum levels of TH17 cytokines are elevated in NMO patientscompared to MS. The TH17 cytokines IL-17A (FIG. 3A) and IL-17F (FIG. 3B)were measured using a luminex multiplex assay. Significance wasdetermined by a student's T-test.

FIGS. 4A-D. IFN-β treatment is therapeutic in TH1-EAE and inflammatoryin TH17-EAE. Mice with TH1-EAE (FIG. 4A) or TH17-EAE (FIG. 4B) weretreated every second day with 1000 units of IFN-β from day 0-day 10 anddiseases scores were monitored until day 45. Inflammation anddemyelination in spinal cords from mice with TH1 (FIG. 4C) and TH17(FIG. 4D) were assessed by staining paraffin embedded sections withhematoxylin and eosin (to assess inflammatory infiltrates) and LuxolFast Blue (to assess myelin integrity).

FIGS. 5A-B. TH17-EAE has severe optic neuritis and visual deficits thanTH1-EAE. (FIG. 5A) Hematoxylin and eosin staining of optic tracks of TH1and TH17-EAE revealed that TH17-EAE had severe inflammation. (FIG. 5B)Optokinetic tracking (visual acuity) is significantly impaired inTH17-EAE compared TH1-EAE.

FIGS. 6A-B. TH17-EAE has increased neutrophils and B-cells in theinflamed spinal cords compared to TH1 EAE. (FIG. 6A) Representative flowcytometry plot of the neutrophils (CD11b+ly6G+), Macrophages(CD11b+F480+), B-cells (CD19+) and T helper cells (CD4+) infiltratingspinal cords of TH1 and TH17-EAE. (FIG. 6B) Mean percentage+/−standarderror of the mean of immune cell populations in the spinal cords of TH1and TH17 EAE. * p<0.05 was determined by a student's T-test.

FIGS. 7A-C. Comparison of the frequency (FIG. 7A) of CD4 T helper cells,(FIG. 7B) Neutrophils and (FIG. 7C) B-cells in the spinal cord, brainand blood from mice with TH1-EAE and TH17-EAE. P-values were determinedusing a Mann-Whitney U test with * p<0.05, **p<0.01, ***p<0.001,

FIG. 8. BAFF and APRIL expression in peripheral blood cells, brain andspinal cords of mice with TH1-EAE or TH17. Statistical analysis wasperformed using a students t-test with p-values *<0.05 and ** <0.01.

FIGS. 9A-B. TH17-EAE spinal chord analysis for BAFF expression. (FIG.9A) Sections from spinal cords of TH17-EAE (clinical score of 3) werestained with anti-BAFF (red) and anti-Ly6G (green) and image on a ZeissLSM-710 confocal microscope. (FIG. 9B) FACS analysis of BAFF expressionfrom subsets of spinal cord infiltrating cells in TH17-EAE (score 3).T-helper cells (CD4+), B-cells (B220+), Macrophage/microglia(CD11b+/GR1), Immature neutrophil/macrophages (CD11b+GR1+), neutrophils(CD11b+/GR1++).

FIG. 10. Cellular and molecular cascade of events that occurs in TH17mediated neuro-inflammation. 1) TH17 cells infiltrate the CNS andsecrete TH17 cytokines (IL-17) which induces the expression ofneutrophil chemokines within the inflamed tissue. 2) Neutrophils migrateto the CNS. 3) Neutrophils express high levels of BAFF and APRIL whichdrives the proliferation of B-cells in the CNS. B-cells then mediatedinflammation by antigen presentation to newly immigrated T-cells and/orby the production of autoantibodies.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventor now provides, for the first time, evidence that mice withTH17-EAE have elevated expression of BAFF and APRIL and increasednumbers of B-cells in inflamed spinal cords and brains as compared tomice with TH1-EAE (FIGS. 1B-D). These data suggest that BAFF, APRIL andB-cells have opposing roles in TH1 and TH17 induced EAE. This leads tothe hypothesis that B-cells are pro-inflammatory in TH17-EAE, but areanti-inflammatory in TH1-EAE. Moreover, the individual functions of BAFFand APRIL in autoimmune diseases also is unclear, and these cytokinesmay have differential effects on inflammation in these two EAE models.These and other aspects of the disclosure are described in detail below.

1. Multiple Sclerosis

Multiple sclerosis (MS), also known as disseminated sclerosis orencephalomyelitis disseminata, is an inflammatory disease in which theinsulating covers of nerve cells in the brain and spinal cord aredamaged. This damage disrupts the ability of parts of the nervous systemto communicate, resulting in a wide range of signs and symptoms,including physical, mental, and sometimes psychiatric problems. MS takesseveral forms, with new symptoms either occurring in isolated attacks(relapsing forms) or building up over time (progressive forms). Betweenattacks, symptoms may disappear completely; however, permanentneurological problems often occur, especially as the disease advances.

While the cause is not clear, the underlying mechanism is thought to beeither destruction by the immune system or failure of themyelin-producing cells. Proposed causes for this include genetics andenvironmental factors such as infections. MS is usually diagnosed basedon the presenting signs and symptoms and the results of supportingmedical tests.

There is no known cure for multiple sclerosis. Treatments attempt toimprove function after an attack and prevent new attacks. Medicationsused to treat MS while modestly effective can have adverse effects andbe poorly tolerated. Many people pursue alternative treatments, despitea lack of evidence. The long-term outcome is difficult to predict, withgood outcomes more often seen in women, those who develop the diseaseearly in life, those with a relapsing course, and those who initiallyexperienced few attacks. Life expectancy is 5 to 10 years lower thanthat of an unaffected population.

As of 2008, between 2 and 2.5 million people are affected globally withrates varying widely in different regions of the world and amongdifferent populations. The disease usually begins between the ages of 20and 50 and is twice as common in women as in men. The name multiplesclerosis refers to scars (sclerae; better known as plaques or lesions)in particular in the white matter of the brain and spinal cord. MS wasfirst described in 1868. A number of new treatments and diagnosticmethods are under development.

A. Disease Manifestations

A person with MS can have almost any neurological symptom or sign; withautonomic, visual, motor, and sensory problems being the most common.The specific symptoms are determined by the locations of the lesionswithin the nervous system, and may include loss of sensitivity orchanges in sensation such as tingling, pins and needles or numbness,muscle weakness, very pronounced reflexes, muscle spasms, or difficultyin moving; difficulties with coordination and balance (ataxia); problemswith speech or swallowing, visual problems (nystagmus, optic neuritis ordouble vision), feeling tired, acute or chronic pain, and bladder andbowel difficulties, among others. Difficulties thinking and emotionalproblems such as depression or unstable mood are also common. Uhthoffsphenomenon, a worsening of symptoms due to exposure to higher than usualtemperatures, and Lhermitte's sign, an electrical sensation that runsdown the back when bending the neck, are particularly characteristic ofMS. The main measure of disability and severity is the expandeddisability status scale (EDSS), with other measures such as the multiplesclerosis functional composite being increasingly used in research.

The condition begins in 85% of cases as a clinically isolated syndromeover a number of days with 45% having motor or sensory problems, 20%having optic neuritis, and 10% having symptoms related to brainstemdysfunction, while the remaining 25% have more than one of the previousdifficulties. The course of symptoms occurs in two main patternsinitially: either as episodes of sudden worsening that last a few daysto months (called relapses, exacerbations, bouts, attacks, or flare-ups)followed by improvement (85% of cases) or as a gradual worsening overtime without periods of recovery (10-15% of cases). A combination ofthese two patterns may also occur or people may start in a relapsing andremitting course that then becomes progressive later on. Relapses areusually not predictable, occurring without warning. Exacerbations rarelyoccur more frequently than twice per year. Some relapses, however, arepreceded by common triggers and they occur more frequently during springand summer. Similarly, viral infections such as the common cold,influenza, or gastroenteritis increase their risk. Stress may alsotrigger an attack. Women with MS who become pregnant experience fewerrelapses; however, during the first months after delivery the riskincreases. Overall, pregnancy does not seem to influence long-termdisability. Many events have not been found to affect relapse ratesincluding vaccination, breast feeding, physical trauma, and Uhthoffsphenomenon.

The three main characteristics of MS are the formation of lesions in thecentral nervous system (also called plaques), inflammation, and thedestruction of myelin sheaths of neurons. These features interact in acomplex and not yet fully understood manner to produce the breakdown ofnerve tissue and in turn the signs and symptoms of the disease.Additionally MS is believed to be an immune-mediated disorder thatdevelops from an interaction of the individual's genetics and as yetunidentified environmental causes. Damage is believed to be caused, atleast in part, by attack on the nervous system by a person's own immunesystem.

1. Lesions

The name multiple sclerosis refers to the scars (sclerae—better known asplaques or lesions) that form in the nervous system. These lesions mostcommonly affect the white matter in the optic nerve, brain stem, basalganglia, and spinal cord, or white matter tracts close to the lateralventricles. The function of white matter cells is to carry signalsbetween grey matter areas, where the processing is done, and the rest ofthe body. The peripheral nervous system is rarely involved.

To be specific, MS involves the loss of oligodendrocytes, the cellsresponsible for creating and maintaining a fatty layer—known as themyelin sheath—which helps the neurons carry electrical signals (actionpotentials). This results in a thinning or complete loss of myelin and,as the disease advances, the breakdown of the axons of neurons. When themyelin is lost, a neuron can no longer effectively conduct electricalsignals. A repair process, called remyelination, takes place in earlyphases of the disease, but the oligodendrocytes are unable to completelyrebuild the cell's myelin sheath. Repeated attacks lead to successivelyless effective remyelinations, until a scar-like plaque is built uparound the damaged axons. These scars are the origin of the symptoms andduring an attack magnetic resonance imaging (MRI) often shows more thanten new plaques. This could indicate that there are a number of lesionsbelow which the brain is capable of repairing itself without producingnoticeable consequences. Another process involved in the creation oflesions is an abnormal increase in the number of astrocytes due to thedestruction of nearby neurons. A number of lesion patterns have beendescribed.

2. Inflammation

Apart from demyelination, the other sign of the disease is inflammation.Fitting with an immunological explanation, the inflammatory process iscaused by T cells, a kind of lymphocyte that plays an important role inthe body's defenses. T cells gain entry into the brain via disruptionsin the blood-brain barrier. The T cells recognize myelin as foreign andattack it, explaining why these cells are also called “autoreactivelymphocytes.”

The attack of myelin starts inflammatory processes, which triggers otherimmune cells and the release of soluble factors like cytokines andantibodies. Further breakdown of the blood-brain barrier, in turn causea number of other damaging effects such as swelling, activation ofmacrophages, and more activation of cytokines and other destructiveproteins. Inflammation can potentially reduce transmission ofinformation between neurons in at least three ways. The soluble factorsreleased might stop neurotransmission by intact neurons. These factorscould lead to or enhance the loss of myelin, or they may cause the axonto break down completely.

B. Diagnosis

Multiple sclerosis is typically diagnosed based on the presenting signsand symptoms, in combination with supporting medical imaging andlaboratory testing. It can be difficult to confirm, especially early on,since the signs and symptoms may be similar to those of other medicalproblems. The McDonald criteria, which focus on clinical, laboratory,and radiologic evidence of lesions at different times and in differentareas, is the most commonly used method of diagnosis with the Schumacherand Poser criteria being of mostly historical significance. While theabove criteria allow for a non-invasive diagnosis, some state that theonly definitive proof is an autopsy or biopsy where lesions typical ofMS are detected.

Clinical data alone may be sufficient for a diagnosis of MS if anindividual has had separate episodes of neurologic symptomscharacteristic of the disease. In those who seek medical attention afteronly one attack, other testing is needed for the diagnosis. The mostcommonly used diagnostic tools are neuroimaging, analysis ofcerebrospinal fluid and evoked potentials. Magnetic resonance imaging ofthe brain and spine may show areas of demyelination (lesions orplaques). Gadolinium can be administered intravenously as a contrastagent to highlight active plaques and, by elimination, demonstrate theexistence of historical lesions not associated with symptoms at themoment of the evaluation. Testing of cerebrospinal fluid obtained from alumbar puncture can provide evidence of chronic inflammation in thecentral nervous system. The cerebrospinal fluid is tested foroligoclonal bands of IgG on electrophoresis, which are inflammationmarkers found in 75-85% of people with MS. The nervous system in MS mayrespond less actively to stimulation of the optic nerve and sensorynerves due to demyelination of such pathways. These brain responses canbe examined using visual- and sensory-evoked potentials.

C. Standard Treatment

Although there is no known cure for multiple sclerosis, severaltherapies have proven helpful. The primary aims of therapy are returningfunction after an attack, preventing new attacks, and preventingdisability. As with any medical treatment, medications used in themanagement of MS have several adverse effects. Alternative treatmentsare pursued by some people, despite the shortage of supporting evidence.

1. Acute Attacks

During symptomatic attacks, administration of high doses of intravenouscorticosteroids, such as methylprednisolone, is the usual therapy, withoral corticosteroids seeming to have a similar efficacy and safetyprofile. Although, in general, effective in the short term for relievingsymptoms, corticosteroid treatments do not appear to have a significantimpact on long-term recovery. The consequences of severe attacks that donot respond to corticosteroids might be treatable by plasmapheresis.

2. Disease-Modifying Treatments Eight disease-modifying treatments havebeen approved by regulatory agencies for relapsing-remitting multiplesclerosis (RRMS) including: interferon β-1a, interferon β-1b, glatirameracetate, mitoxantrone, natalizumab, fingolimod, teriflunomide anddimethyl fumarate. Their cost effectiveness is unclear.

In RRMS they are modestly effective at decreasing the number of attacks.The interferons and glatiramer acetate are first-line treatments and areroughly equivalent, reducing relapses by approximately 30%.Early-initiated long-term therapy is safe and improves outcomes.Natalizumab reduces the relapse rate more than first-line agents;however, due to issues of adverse effects is a second-line agentreserved for those who do not respond to other treatments or with severedisease. Mitoxantrone, whose use is limited by severe adverse effects,is a third-line option for those who do not respond to othermedications. Treatment of clinically isolated syndrome (CIS) withinterferons decreases the chance of progressing to clinical MS. Efficacyof interferons and glatiramer acetate in children has been estimated tobe roughly equivalent to that of adults. The role of some of the neweragents such as fingolimod, teriflunomide, and dimethyl fumarate, as of2011, is not yet entirely clear.

No treatment has been shown to change the course of primary progressiveMS and as of 2011 only one medication, mitoxantrone, has been approvedfor secondary progressive MS. In this population tentative evidencesupports mitoxantrone moderately slowing the progression of the diseaseand decreasing rates of relapses over two years.

The disease-modifying treatments have several adverse effects. One ofthe most common is irritation at the injection site for glatirameracetate and the interferons (up to 90% with subcutaneous injections and33% with intramuscular injections). Over time, a visible dent at theinjection site, due to the local destruction of fat tissue, known aslipoatrophy, may develop. Interferons may produce flu-like symptoms;some people taking glatiramer experience a post-injection reaction withflushing, chest tightness, heart palpitations, breathlessness, andanxiety, which usually lasts less than thirty minutes. More dangerousbut much less common are liver damage from interferons, systolicdysfunction (12%), infertility, and acute myeloid leukemia (0.8%) frommitoxantrone, and progressive multifocal leukoencephalopathy occurringwith natalizumab (occurring in 1 in 600 people treated).

Both medications and neurorehabilitation have been shown to improve somesymptoms, though neither changes the course of the disease. Somesymptoms have a good response to medication, such as an unstable bladderand spasticity, while others are little changed. For neurologicproblems, a multidisciplinary approach is important for improvingquality of life; however, it is difficult to specify a ‘core team’ asmany different health services may be needed at different points intime. Multidisciplinary rehabilitation programs increase activity andparticipation of people with MS but do not influence impairment level.There is limited evidence for the overall efficacy of individualtherapeutic disciplines, though there is good evidence that specificapproaches, such as exercise, and psychology therapies, in particularcognitive behavioral approaches are effective.

Over 50% of people with MS may use complementary and alternativemedicine, although percentages vary depending on how alternativemedicine is defined. The evidence for the effectiveness for suchtreatments in most cases is weak or absent. Treatments of unprovenbenefit used by people with MS include: dietary supplementation andregimens, vitamin D, relaxation techniques such as yoga, herbal medicine(including medical cannabis), hyperbaric oxygen therapy, self-infectionwith hookworms, reflexology and acupuncture. Regarding thecharacteristics of users, they are more frequently women, have had MSfor a longer time, tend to be more disabled and have lower levels ofsatisfaction with conventional healthcare.

3. Side Effects

Fingolimod may give rise to hypertension and bradycardia, macular edema,elevated liver enzymes or a reduction in lymphocyte levels. Tentativeevidence supports the short term safety of teriflunomide, with commonside effects including: headaches, fatigue, nausea, hair loss, and limbpain. There have also been reports of liver failure and PML with its useand it is dangerous for fetal development. Most common side effects ofdimethyl fumarate are flushing and gastrointestinal problems. Whiledimethyl fumarate may lead to a reduction in the white blood cell countthere were no reported cases of opportunistic infections during trials.

II. NEUROMYELITIS OPTICA

Neuromyelitis optica (NMO), also known as Devic's disease or Devic'ssyndrome, is an autoimmune, inflammatory disorder that attacks the opticnerves and spinal cord. This produces an inflammation of the optic nerve(optic neuritis) and the spinal cord (myelitis). Although inflammationmay also affect the brain, the lesions are different from those observedin the related condition multiple sclerosis (MS). Spinal cord lesionslead to varying degrees of weakness or paralysis in the legs or arms,loss of sensation (including blindness), and/or bladder and boweldysfunction. NMO is a rare disorder, which resembles MS in several ways,but requires a different course of treatment for optimal results. Alikely target of the autoimmune attack at least in some patients withNMO is a protein of the nervous system cells called aquaporin 4.

NMO is similar to MS in that the body's immune system attacks the myelinsurrounding nerve cells. Unlike standard MS, the attacks are notbelieved to be mediated by the immune system's T cells but rather byantibodies called NMO-IgG, or simply NMO antibodies. These antibodiestarget a protein called aquaporin 4 in the cell membranes of astrocytes,which acts as a channel for the transport of water across the cellmembrane. Aquaporin 4 is found in the processes of the astrocytes thatsurround the blood-brain barrier, a system responsible for preventingsubstances in the blood from crossing into the brain. The blood-brainbarrier is weakened in NMO, but it is currently unknown how the NMO-IgGimmune response leads to demyelination.

A. Disease Manifestations

The main symptoms of NMO are loss of vision and spinal cord function. Asfor other etiologies of optic neuritis, the visual impairment usuallymanifests as decreased visual acuity, although visual field defects, orloss of color vision may occur in isolation or prior to formal loss ofacuity. Spinal cord dysfunction can lead to muscle weakness, reducedsensation, or loss of bladder and bowel control. The typical patient hasan acute and severe spastic weakness of the legs (paraparesis) or allfour limbs (tetraparesis) with sensory signs, often accompanied by lossof bladder control.

As discussed above, NMO is similar to MS in that the body's immunesystem attacks the myelin surrounding nerve cells. Unlike standard MS,the attacks are not believed to be mediated by the immune system's Tcells but rather by antibodies called NMO-IgG, or simply NMO antibodies.These antibodies target a protein called aquaporin 4 in the cellmembranes of astrocytes, which acts as a channel for the transport ofwater across the cell membrane. Aquaporin 4 is found in the processes ofthe astrocytes that surround the blood-brain barrier, a systemresponsible for preventing substances in the blood from crossing intothe brain. The blood-brain barrier is weakened in NMO, but it iscurrently unknown how the NMO-IgG immune response leads todemyelination.

Most research into the pathology of NMO has focused on the spinal cord.The damage in the spinal cord can range from inflammatory demyelinationto necrotic damage of the white and grey matter. The inflammatorylesions in NMO have been classified as type II lesions (complementmediated demyelinization), but they differ from MS pattern II lesions intheir prominent perivascular distribution. Therefore, the pattern ofinflammation is often quite distinct from that seen in MS.

Approximately 20% of patients with monophasic NMO have permanent visualloss and 30% have permanent paralysis in one or more legs. Amongpatients with relapsing NMO, 50% have paralysis or blindness within 5years. In some patients (33% in one study), transverse myelitis in thecervical spinal cord resulted in respiratory failure and subsequentdeath. However, the spectrum of NMO has widened due to improveddiagnostic criteria, and the options for treatment have improved; as aresult, researchers believe that these estimates will be lowered.

The prevalence and incidence of NMO has not been established partlybecause the disease is underrecognized and often confused with MS. NMOis more common in women than men, with women comprising over 2/3 ofpatients and more than 80% of those with the relapsing form of thedisease. NMO is more common in Asiatic people than Caucasians. In fact,Asian optic-spinal MS (which constitutes 30% of the cases of MS inJapan) has been suggested to be identical to NMO (differences betweenoptic-spinal and classic MS in Japanese patients). In the indigenouspopulations of tropical and subtropical regions, MS is rare, but when itappears it often takes the form of optic-spinal MS. The majority of NMOpatients have no affected relatives, and it is generally regarded as anon-familial condition.

B. Traditional Diagnosis

The Mayo Clinic proposed a revised set of criteria for diagnosis of NMOin 2006. The new guidelines for diagnosis require two absolute criteriaplus at least two of three supportive criteria being:

Absolute Criteria:

-   -   Optic neuritis    -   Acute myelitis

Supportive Criteria:

-   -   Brain MRI not meeting criteria for MS at disease onset    -   Spinal cord MRI with contiguous T2-weighted signal abnormality        extending over 3 or more vertebral segments, indicating a        relatively large lesion in the spinal cord

NMO-IgG Seropositive Status:

-   -   The NMO-IgG test checks the existence of antibodies against the        aquaporin 4 antigen        After the development of the NMO-IgG test, the spectrum of        disorders that comprise NMO was expanded. The NMO spectrum is        now believed to consist of:    -   Standard NMO, according to the diagnostic criteria described        above    -   Limited forms of NMO, such as single or recurrent events of        longitudinally extensive myelitis, and bilateral simultaneous or        recurrent optic neuritis    -   Asian optic-spinal MS. This variant can present CNS involvement        like MS    -   Longitudinally extensive myelitis or optic neuritis associated        with systemic auto-immune disease    -   Optic neuritis or myelitis associated with lesions in specific        brain areas such as the hypothalamus, periventricular nucleus,        and brainstem        Whether NMO is a distinct disease or part of the wide spectrum        of multiple sclerosis is debated. Recently it has been found        that antiviral immune response distinguishes MS and NMO, but        being MS an heterogeneous condition, as hepatitis or diabetes        are, it is still possible to consider NMO part of the MS        spectrum.

NMO has been associated with many systemic diseases, based on anecdotalevidence of some NMO patients with a comorbid condition. Such conditionsinclude: collagen vascular diseases, autoantibody syndromes, infectionswith varicella-zoster virus, Epstein-Barr virus, and HIV, and exposureto clioquinol and antituberculosis drugs.

C. Therapy and Prophylaxis

It may be that, on the basis of the diagnosis or prediction provided bythe methods described herein, one will wish to begin, end or modify atherapeutic regimen. In particular, subjects diagnosed as having or atrisk of developing NMO may be started on a therapeutic regimen. Theprimary aims of therapy are returning function after an attack,preventing new attacks, and preventing disability. As with any medicaltreatment, medications used in the management of NMO have severaladverse effects, and many possible therapies are still underinvestigation.

Currently, there is no cure for NMO, but symptoms can be treated. Somepatients recover, but many are left with impairment of vision and limbs,which can be severe. Attacks are treated with short courses of highdosage intravenous corticosteroids such as methylprednisolone IV. Whenattacks progress or do not respond to corticosteroid treatment,plasmapheresis can be an effective treatment. Clinical trials for thesetreatments contain very small numbers, and most are uncontrolled.

No controlled trials have established the effectiveness of treatmentsfor the prevention of attacks. Many clinicians agree that long-termimmunosuppression is required to reduce the frequency and severity ofattacks, while others argue the exact opposite. Commonly usedimmunosuppressant treatments include azathioprine (Imuran) plusprednisone, mycophenolate mofetil plus prednisone, Rituximab,Mitoxantrone, intravenous immunoglobulin (IVIG), and Cyclophosphamide.The monoclonal antibody rituximab is under study. In 2007, NMO wasreported to be responsive to glatiramer acetate and to low-dosecorticosteroids. Normally, there is some measure of improvement in a fewweeks, but residual signs and disability may persist, sometimesseverely.

The disease can be monophasic, i.e., a single episode with permanentremission. However, at least 85% of patients have a relapsing form ofthe disease with repeated attacks of transverse myelitis and/or opticneuritis. In patients with the monophasic form the transverse myelitisand optic neuritis occur simultaneously or within days of each other. Onthe other hand, patients with the relapsing form are more likely to haveweeks or months between the initial attacks and to have better motorrecovery after the initial transverse myelitis event. Relapses usuallyoccur early with about 55% of patients having a relapse in the firstyear and 90% in the first 5 years. Unlike multiple sclerosis, NMO rarelyhas a secondary progressive phase in which patients have increasingneurologic decline between attacks without remission. Instead,disabilities arise from the acute attacks.

III. TREATMENTS

In accordance with the present disclosure, therapeutic interventionsinto MS and NMO are provided. The inventor has identified theinvolvement of TH17-induced disease in certain types of MS, and foundthat these subjects are characterized by non-response to interferontherapy. NMO exhibits a similar TH17-driven disease profile.Furthermore, both of these diseases appear to be linked to increasedlevels of B-cell activating factor (BAFF) and proliferation-inducingligand (APRIL). While these proteins have been shown to be viabletherapeutic targets in systemic lupus erythematosus, attempts to targetthem in MS have failed. However, with a better understanding of thediffering cytokine responses in MS subtypes, BAFF/APRIL-targetedtherapies may indeed succeed, and are certainly applicable to NMO.

A. BAFF

B-cell activating factor (BAFF) also known as tumor necrosis factorligand superfamily member 13B is a cytokine that in humans is encoded bythe TNFSF13B gene. BAFF is also known as B Lymphocyte Stimulator (BLyS)and TNF- and APOL-related leukocyte expressed ligand (TALL-1) and theDendritic cell-derived TNF-like molecule (CD257 antigen; cluster ofdifferentiation 257). This cytokine is a ligand for receptorsTNFRSF13B/TACI, TNFRSF17/BCMA, and TNFRSF13C/BAFF-R. This cytokine isexpressed in B cell lineage cells, and acts as a potent B cellactivator. It has been also shown to play an important role in theproliferation and differentiation of B cells. Excessive level of BAFFcauses abnormally high antibody production, results in systemic lupuserythmatosis, rheumatoid arthritis, and many other autoimmune diseases.

BAFF is a 285-amino acid long peptide glycoprotein which undergoesglycosylation at residue 124. It is expressed as a membrane-bound typeII transmembrane protein on various cell types including monocytes,dendritic cells and bone marrow stromal cells. The transmembrane formcan be cleaved from the membrane, generating a soluble protein fragment.BAFF steady-state concentrations depend on B cells and also on theexpression of BAFF-binding receptors. BAFF is the natural ligand ofthree unusual tumor necrosis factor receptors named BAFF-R (BR3), TACI(transmembrane activator and calcium modulator and cyclophilin ligandinteractor), and BCMA (B-cell maturation antigen), all of which havediffering binding affinities for it. These receptors are expressedmainly on mature B lymphocytes and their expression varies in dependenceof B cell maturation (TACI is also found on a subset of T-cells and BCMAon plasma cells). BAFF-R is involved in the positive regulation during Bcell development. TACI binds worst since its affinity is higher for aprotein similar to BAFF, called a proliferation-inducing ligand (APRIL).BCMA displays an intermediate binding phenotype and will work witheither BAFF or APRIL to varying degrees. Signaling through BAFF-R andBCMA stimulates B lymphocytes to undergo proliferation and to counterapoptosis. All these ligands act as homotrimers (i.e. three of the samemolecule) interacting with homotrimeric receptors, although BAFF hasbeen known to be active as either a hetero- or homotrimer (can aggregateinto 60-mer depending on the primary structure of the protein).

B-cell activating factor has been shown to interact with TNFRSF13B,TNFSF13 and TNFRSF17. Interaction between BAFF and BAFF-R activatesclassical and noncanonical NF-κB signaling pathways. This interactiontriggers signals essential for the formation and maintenance of B cell,thus it is important for a B-cell survival.

As an immunostimulant, BAFF (BLyS, TALL-1) is necessary for maintainingnormal immunity. Inadequate level of BAFF will fail to activate B cellsto produce enough immunoglobulin and will lead to immunodeficiency.

Belimumab (Benlysta) is a monoclonal antibody developed by Human GenomeSciences and GlaxoSmithKline, with significant discovery input byCambridge Antibody Technology, which specifically recognizes andinhibits the biological activity of B-Lymphocyte stimulator (BLyS) andis in clinical trials for treatment of Systemic lupus erythematosus andother auto-immune diseases.

BAFF has been found in renal transplant biopsies with acute rejectionand correlate with appearance C4d. Increased levels of BAFF may initiatealoreactive B cell and T cell immunity, therefore may promote allograftrejection. Lower levels of BAFF transcripts (or higher levels of solubleBAFF) show a higher risk of producing donor-specific antibodies in theinvestigated patients. Donor-specific antibodies bind with high affinityto the vascular endothelium of graft and activate complement. Thisprocess results in neutrophils infiltration, hemorrhage, fibrindeposition and platelet aggregation. Targeting BAFF-R interactions mayprovide new therapeutic possibilities in transplantation.

Blisibimod, a fusion protein inhibitor of BAFF, is in development byAnthera Pharmaceuticals, also primarily for the treatment of systemiclupus erythematosus.

B. April

Tumor necrosis factor ligand superfamily member 13 (TNFSF13) also knownas a proliferation-inducing ligand (APRIL) is a protein that in humansis encoded by the TNFSF13 gene. TNFSF13 has also been designated CD256(cluster of differentiation 256).

The protein encoded by this gene is a member of the tumor necrosisfactor ligand (TNF) ligand family. This protein is a ligand forTNFRSF17/BCMA, a member of the TNF receptor family. This protein and itsreceptor are both found to be important for B cell development. In vivoexperiments suggest an important role for APRIL in the long-termsurvival of plasma cells in the bone marrow. Mice deficient in Aprilshowed a reduced ability to support plasma cell survival In vitroexperiments suggested that this protein may be able to induce apoptosisthrough its interaction with other TNF receptor family proteins such asTNFRSF6/FAS and TNFRSF14/HVEM. Three alternatively spliced transcriptvariants of this gene encoding distinct isoforms have been reported.TNFSF13 has been shown to interact with TNFRSF13B and B-cell activatingfactor.

C. Pharmaceutical Compositions

1. Therapeutic Agents

The present disclosure envisions using variety of BAFF and APRILinhibitors. In general, inhibitors are biological in nature (proteins,nucleic acids, carbohydrates) or synthetic (small molecules, peptoids,nucleic acid analogs, etc.). The former includes antibodies to BAFF orAPRIL, and nucleic acid inhibitors (siRNAs; antisense molecules) thatcan be readily produced by those of skill in the art by design orpreparation based on the targets themselves. The following are a fewexamples of biological inhibitors.

Atacicept.

Atacicept is a recombinant fusion protein designed to inhibit B cells,thereby suppressing autoimmune disease. The designer protein combinesthe binding site for two cytokines that regulate maturation, function,and survival of B cells, B-lymphocyte stimulator (BLyS) and aproliferation-inducing ligand (APRIL), with the constant region ofimmunoglobin. Atacicept blocks activation of B cells by the tumornecrosis factor receptor superfamily member 13B (more commonly known asTACI), a transmembrane receptor protein found predominantly on thesurface of B cells. Like the monoclonal antibody belimumab, ataciceptblocks the binding of BLyS, but it also blocks APRIL. Binding of theseTACI ligands induces proliferation, activation, and longevity of B cellsand thus their production of autoantibodies. Atacicept is thought toselectively impair mature B cells and plasma cells with less impact onprogenitor cells and memory B cells.

Studies have looked at atacicept in animal models of autoimmune diseaseand in patients with systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), and optic neuritis. A phase II/III trial for systemiclupus erythematosus is due to run from 2008 to 2012. The subcutaneouslyinjected protein failed a phase II trial for multiple sclerosis. Thetrials of atacicept in people with MS were suspended when some peopletaking the drug in one trial had an unexpected increase in inflammatoryactivity. An independent data monitoring board for the MS study found“subjects receiving atacicept were having more relapses and new MRIlesions than those on the placebo.” The drug is also being studied fortreatment of B-cell malignancies, including multiple myeloma, B-cellchronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

Belimumab.

Belimumab (trade name Benlysta) is a human monoclonal antibody thatinhibits B-cell activating factor (BAFF), also known as B-lymphocytestimulator (BLyS). B cells are responsible for part of the normal immuneresponse, and also for the over-aggressive immune response in autoimmunediseases like systemic lupus erythematosus (SLE). Belimumab reduces thenumber of circulating B cells. It is possible that belimumab bindsprimarily to circulating soluble BAFF, therefore not inducingantibody-dependent cellular cytotoxicity that could be expected fromthis IgG1-type antibody.

Belimumab is approved in the United States, Canada and Europe fortreatment of SLE. However, the major phase III trials excluded the moresevere cases of SLE with kidney and brain damage, so its effectivenesshas not been demonstrated in those cases. A Phase III study for SLEpatients with kidney disease is now recruiting. U.S. FDA reviewers wereconcerned that belimumab is only “marginally” effective, and that therewere more deaths in the treatment group. Belimumab's defenders said thatin addition to its modest efficiency, belimumab allowed patients tosignificantly reduce their use of corticosteroids. Phase II trials ofbelimumab for rheumatoid arthritis were unsuccessful. Phase II trialsfor Sjögren's Syndrome were more successful.

While belimumab appears safe in systemic lupus erythematosus, themagnitude of benefit is small. Black/African American patients did notshow a benefit. The most severe cases, with kidney and central nervoussystem involvement, were excluded from the trials.

The efficacy and safety of belimumab was demonstrated in 2 Phase IIIrandomized, controlled studies, BLISS-52 and BLISS-76. The 2 studies hada total of 1,684 patients, with scores of ≧6 on the SELENA-SLEDAIassessment. They were divided into a placebo and 2 dosage groups ofbelimumab, in addition to standard therapy. The primary end point was areduction of ≧4 on the SELENA-SLEDAI assessment, and several otherfactors, at 52 weeks. Belimumab significantly improved the responserate, reduced disease activity and severe flares, and was welltolerated. 58% had SELENA-SLEDAI scores reduced by ≧4 points during 52weeks with belimumab 10 mg/kg compared to 46% with placebo.

Common adverse effects reported with belimumab include nausea, diarrhea,fever, as well as hypersensitivity and infusion-site reactions (severein 0.9% of patients). It is suggested that patients be treated with anantihistamine prior to a belimumab infusion. A greater number of seriousinfections and deaths were reported in patients treated with belimumabthan in those treated with placebo. Infections are due to theimmunosuppressant properties of the drug.

Blisibimod.

Blisibimod (also known as A-623, formerly AMG 623) is a selectiveantagonist of BAFF, being developed by Anthera Pharmaceuticals as atreatment for systemic lupus erythematosus. It is currently under activeinvestigation in clinical trials. It is a fusion protein consisting offour BAFF binding domains fused to the N-terminus of the fragmentcrystallizable region (Fc) of a human antibody.

Tabalumab.

Tabalumab (LY 2127399) is an anti-B-cell activating factor (BAFF) humanmonoclonal antibody designed for the treatment of autoimmune diseasesand B cell malignancies. Tabalumab was developed by Eli Lilly andCompany. A phase III clinical trial for rheumatoid arthritis was haltedin February of 2013.

2. Formulations and Routes of Administration

Where therapeutic applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. Generally, this will entail preparing compositionsthat are essentially free of pyrogens, as well as other impurities thatcould be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery agents stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the agent, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrases “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the agents of the present invention, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. Such routes includeoral, nasal, buccal, rectal, vaginal or topical route. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Inthis connection, sterile aqueous media which can be employed will beknown to those of skill in the art in light of the present disclosure.For example, one dosage could be dissolved in 1 ml of isotonic NaClsolution and either added to 1000 ml of hypodermoclysis fluid orinjected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences,” 15^(th) Ed., 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

D. Combination Therapies

It is common in many fields of medicine to treat a disease with multipletherapeutic modalities, often called “combination therapies.” MS and NMOmay benefit from such an approach as well. To treat these disordersusing the methods and compositions of the present disclosure, one wouldgenerally contact a subject with an agent according to the presentdisclosure and at least one other therapy. These therapies would beprovided in a combined amount effective to achieve a reduction in one ormore disease parameter. This process may involve contacting the subjectwith the both agents/therapies at the same time, e.g., using a singlecomposition or pharmacological formulation that includes both agents, orby contacting the subject with two distinct compositions orformulations, at the same time, wherein one composition includes theagents of the present disclosure and the other includes the otheragent/therapy.

Alternatively, the agent of the present disclosure may precede or followthe other agent/treatment by intervals ranging from minutes to weeks.One would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the therapies wouldstill be able to exert an advantageously combined effect on thecell/subject. In such instances, it is contemplated that one wouldcontact the cell with both modalities within about 12-24 hours of eachother, within about 6-12 hours of each other, or with a delay time ofonly about 12 hours. In some situations, it may be desirable to extendthe time period for treatment significantly; however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

It also is conceivable that more than one administration of either theagent of the present disclosure or the other therapy will be desired.Various combinations may be employed, where the agent of the presentdisclosure is “A,” and the other therapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated. The following discussion lists someagents that can be used as the “other therapy” in combination withagents of the present disclosure.

Elastase inhibitors find use in the treatment of NMO and IL-17-type MS.Inhibitors are known in the art, and include without limitation,sivelestat sodium hydrate (Ono Pharmaceutical); alphal-antitrypsin;pafistatin-like protease inhibitors (de Marco, 2010), Peptides31(7):1280-1260); Clitocybin D (Kim et al., 2009, J MicrobiolBiotechnol. 19(10):1139-41); marama bean inhibitor (Nadaraja et al.,2010, J Enzyme Inhib Med Chem. 25(3):377-82; AE-3763 (Inoue et al.,2009, Bioorg Med Chem. 17(21):7477-86); Isodeoxyhelicobasidin (Xu etal., 2009, J Antibiot 62(6):333-4); guamerin (Jo et al., 2008, IntImmunopharmacol. 8(7):959-66); elafin (Wang et al., 2008, Am J RespirCell Mol Biol. 38(6):724-32); Bornyl (3,4,5-trihydroxy)-cinnamate(Steinbrecher et al., 2008, Bioorg Med Chem. 16(5):2385-90); and thelike as known in the art. Other inhibitors of interest includeantibodies specific for neutrophil elastase, anti-senseoligonucleotides, siRNA, shRNA, and the like.

Gro-alpha inhibitors are also of interest, e.g., antileukinate (see, forexample Fujisawa et al. (1999) Melanoma res. 9(2):105-114. Otherinhibitors of interest include antibodies specific for gro-alpha,anti-sense oligonucleotides, siRNA, shRNA, and the like.

In one embodiment, modulators of T cell and/or granulocyte activity areused in the treatment of inflammatory demyelinating disease of an IL-17subtype, including subtypes of MS and NO. Patients may be classifiedaccording to cytokine subtype prior to administration of a granulocyteinhibitor, particularly MS patients. NO patients generally have an IL-17type disease, and may be treated with a granulocyte inhibitor in theabsence of cytokine profiling.

In some embodiments, the therapeutic agent is an elastase inhibitor,e.g., a small molecule inhibitor which may include without limitation,sivelestat sodium hydrate; α1-antitrypsin; pafistatin-like proteaseinhibitors; Clitocybin D); marama bean inhibitor; AE-3763;Isodeoxyhelicobasidin; guamerin; elafin; Bornyl(3,4,5-trihydroxy)-cinnamate; and the like as known in the art.Alternatively the therapeutic agent is an inhibitor of gro-alpha, e.g.,peptides, small molecules, and the like.

In some embodiments, the other agents are antibodies specific for agranulocyte marker, e.g., elastase, gro-alpha, etc. The term “antibody”is used in the broadest sense and specifically covers monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desired biologicalactivity. “Antibodies” (Abs) and “immunoglobulins” (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which lack antigen specificity. Polypeptides of the latterkind are, for example, produced at low levels by the lymph system and atincreased levels by myelomas.

Corticosteroids have a short onset of action. Other disease modifyingdrugs take several weeks or months to demonstrate a clinical effect.These agents include methotrexate, leflunomide (Arava™), etanercept(Enbrel™), infliximab (Remicade™), adalimumab (Humira™), anakinra(Kineret™), rituximab (Rituxan™), CTLA4-Ig (abatacept), antimalarials,gold salts, sulfasalazine, d-penicillamine, cyclosporin A,cyclophosphamide azathioprine; and the like.

Antigen specific therapeutic methods include administration of anantigen or epitope specific therapeutic agent. One method to induceimmune tolerance is tolerizing DNA vaccines (Garren et al., 2001,Immunity, 15:15-22; Robinson et al., 2003, Nature Biotechnology21:1033-9). Tolerizing DNA vaccines are DNA plasmids containing theregulatory regions necessary for expression of the encoded cDNA inmammalian cells, and would be engineered to contain cDNA sequenceencoding all or a portion of a targeted antigen in order to induceimmune tolerance to the encoded epitopes. To enhance the ability of suchplasmids to induce immune tolerance, the immunostimulatory CpG sequences(Krieg et al., 1998, Trends Microbiol. 6:23-27) can be reduced in numberor completely removed from the plasmid vector. Additionally,immunoinhibitory GpG sequences can be added to the vector (see Ho etal., 2005, J. Immunology, 175:6226-34). Tolerizing DNA plasmids aredelivered intramuscularly to induce immune tolerance to an antigen,thereby reducing T cell and autoantibody responses to reduce autoimmunedestruction of the myelin sheath.

As an alternative, or in addition to DNA tolerization, specificpeptides, altered peptides, or proteins may be administeredtherapeutically to induce antigen-specific tolerance to treatautoimmunity. Native peptides targeted by the autoimmune response can bedelivered to induce antigen-specific tolerance (Gaur et al., Science258:1491-4, 1992). Native peptides have been delivered intravenously toinduce immune tolerance (Warren et al., J. Neurol. Sci. 152:31-8, 1997).Delivery of peptides that are altered from the native peptide, is alsoknown in the art. Alteration of native peptides with selective changesof crucial residues (altered peptide ligands or “APL”) can induceunresponsiveness or change the responsiveness of antigen-specificautoreactive T cells. In another embodiment, whole protein antigenstargeted by the autoimmune response can be delivered to restore immunetolerance to treat autoimmunity (Critchfield et al., Science 263:1139,1994).

E. Companion Diagnostics

In another aspect, the disclosure contemplates the application ofcompanion diagnostic testing to determine the applicability ofBAFF/APRIL-targeted therapies, as well as the inapplicability ofinterferon-based therapies. The inventor has determined that Th17-drivendisease states (NMO, some MS) will not respond to interferon therapy andshould respond favorably to BAFF/APRIL-targeted therapies. Thus,determining the levels of BAFF and/or APRIL in subjects will proveuseful in guiding therapeutic options for these patients.

Assessment of expression levels of may be direct, as in the use ofquantitative immunohistochemistry (IHC) or other antibody-based assays(Western blot, FIA, FISH, radioimmunoassay (RIA), RIP, ELISA,immunoassay, immunoradiometric assay, a fluoroimmunoassay, animmunoassay, a chemiluminescent assay, a bioluminescent assay, a gelelectrophoresis), or indirectly by quantitating the transcripts forthese genes (in situ hybridization, nuclease protection, Northern blotor PCR, including RT-PCR). Relevant methodologies are discussed below.

1. Nucleic Acid-Based Methods

The present invention comprises methods of examining mRNA expression asa measure of target protein levels. mRNA is isolated from cancer cellsaccording to standard methodologies (Sambrook et al., 1989). It may bedesired to convert the RNA to a complementary DNA. In one embodiment,the RNA is whole cell RNA; in another, it is poly-A RNA. Normally, thenucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

A variety of different assays are contemplated, including but notlimited to, fluorescent in situ hybridization (FISH), Northern blotting,dot blot analysis, and of course PCR and RT-PCR. A number of templatedependent processes are available to amplify the marker sequencespresent in a given template sample. One of the best known amplificationmethods is the polymerase chain reaction (referred to as PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, and in Innis et al., 1990, each of which is incorporatedherein by reference in its entirety.

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each technique providesdifferent types of information, although cDNA blotting is analogous, inmany aspects, to blotting or RNA species.

Briefly, a probe is used to target a RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by “blotting” on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols. See Sambrook et al. (1989).For example, chromophore or radiolabel probes or primers identify thetarget during or following amplification.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences that may then be analyzed by direct sequencing.

2. Immunodiagnostics

Antibodies of can be used in characterizing the survivin content oftarget cells through techniques such as ELISAs and Western blotting. Theuse of antibodies in the present invention in an ELISA assay iscontemplated. For example, antibodies are immobilized onto a selectedsurface, preferably a surface exhibiting a protein affinity such as thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed material, it is desirable to bind or coat theassay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

After binding of antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the sample to be tested in amanner conducive to immune complex (antigen/antibody) formation.

Following formation of specific immunocomplexes between the test sampleand the bound antibody, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for a tumor suppressor thatdiffers the first antibody. Appropriate conditions preferably includediluting the sample with diluents such as BSA, bovine gamma globulin(BGG) and phosphate buffered saline (PBS)/Tween®. These added agentsalso tend to assist in the reduction of nonspecific background. Thelayered antisera is then allowed to incubate for from about 2 to about 4hr, at temperatures preferably on the order of about 25° to about 27° C.Following incubation, the antisera-contacted surface is washed so as toremove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween®, or borate buffer.

To provide a detecting means, the second antibody will preferably havean associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease or peroxidase-conjugated anti-human IgG for aperiod of time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

The preceding format may be altered by first binding the sample to theassay plate. Then, primary antibody is incubated with the assay plate,followed by detecting of bound primary antibody using a labeled secondantibody with specificity for the primary antibody.

Antibodies can also find use in immunoblots or Western blot analysis.The antibodies may be used as high-affinity primary reagents for theidentification of proteins immobilized onto a solid support matrix, suchas nitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground Immunologically-based detection methods for use inconjunction with Western blotting include enzymatically-, radiolabel-,or fluorescently-tagged secondary antibodies against the tumorsuppressor are considered to be of particular use in this regard.

F. Kits

For use in the applications described herein, kits are also within thescope of the disclosure. Such kits can comprise a carrier, package orcontainer that is compartmentalized to receive one or more containerssuch as vials, tubes, and the like, each of the container(s) comprisingone of the separate elements to be used in the methods disclosed herein.The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial end user standpoint, including buffers, diluents,filters, and package inserts with instructions for use. In addition, alabel can be provided on the container to indicate that the compositionis used for a specific therapeutic or diagnostic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit. In particular,kits according to the present invention contemplate the assemblage ofagents for assessing leves of the biomarkers discussed above along withone or more of a therapeutic and/or a reagent, as well as controls forassessing the same.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Materials and Methods

Induction of TH1- and TH17-EAE.

To induce TH1- and TH17 EAE, the inventor will immunize donor C57BL/6 orSJL mice with MOG₃₅₋₅₅ or PLP₁₃₉₋₁₅₁ peptides, respectively, in completeFreund's adjuvant. Ten days post immunization, spleens and lymph nodesfrom these mice will be harvested and cultured for 3 days with theimmunizing antigen in conditions favoring TH1 (IL-12 and anti-IL-4) orTH17 (IL-23, anti-IFγ and anti-IL-4) differentiation. After culturing,5-10×10⁶ cells from each condition will be injected intravenously intohealthy recipient mice of the same background. Paralysis follows thetypical EAE progression and mice will be monitored and scored asfollows: 0, normal; 1, tail paralysis; 2, hind limb weakness; 3,complete hind limb paralysis; 4, hind limb paralysis with forelimbweakness and 5, moribund or death (see FIG. 1A).

Assess the Dynamics of BAFF and APRIL Expression.

The first goal of this objective is to assess the expression of BAFF andAPRIL throughout the course of TH1 and TH17-EAE. The inventor hasalready shown that the transcription of BAFF and APRIL genes areelevated in the spinal cords of TH17-EAE compared to TH1-EAE (see FIG.1B). However, both BAFF and APRIL are expressed as membrane boundmolecules which become activated upon cleavage with furin-likeproteases. Therefore, the inventor will measure protein expression ofBAFF and APRIL in spleens, serum, brain and spinal cord by western blot.This will allow us to confirm that the protein is indeed translated inthese tissues during disease and also assess the ratio of membrane bound(inactive) and cleaved (active) forms of these molecules.

Characterization of B-Cell Subsets in TH1 and TH17 EAE.

The second goal of this objective is to assess the tissue distributionof B-cell subsets during TH1 and TH17 EAE. The inventor will phenotypeB-cell subsets in blood, spleen, lymph nodes, and central nervous system(CNS) at four time points during TH17 and TH1-EAE. The time points willbe prior to onset of paralysis, at the time of disease onset, at thepeak of disease and during the chronic phase of the disease. B-cellsubsets will be identified by flow cytometry with the following scheme.Newly developed B-cells will be defined as CD19⁺IgM^(high)IgD^(low)CD93⁺, mature follicular B-cells asCD19⁺IgM^(low)IgD⁺CD1d^(low)CD21/35^(low); regulatory B-cells asCD19⁺CD1d^(high)CD21^(high)IgM^(high)IgD^(+/−)CD5⁺; germinal centerB-cells as CD19⁺PNA⁺GL7⁺; and plasmablasts/cells asB220^(+/−)CD138⁺CD267⁺. In addition, the inventor will assess thedevelopment of anti-myelin antibodies throughout the course of EAE byELISA.

Statistical Analysis.

Results from these experiments will be presented as means+/−one standarddeviation, and significance will be determined by a two-tailed Student'st test or one way ANOVA.

Treatment of TH1 and TH17 EAE with TACI-Ig and Anti-APRIL.

In this study, the inventors presents data (n=3 mice/group) showing thatintraperitoneal injections of TACI-Ig (100 μg/dose every 3^(rd) day)reverses the clinical course of TH17-EAE when treatment is initiated atthe peak of acute disease (see FIG. 2A). The inventor will expand thiswork to include TACI-Ig and anti-APRIL to treat both TH1 and TH17 EAEmodels. In addition, the inventor will use PBS, human IgG (control forTACI-Ig) and mouse IgG2b (control for anti-APRIL) as appropriatetreatment controls. Treatments (100 μg of treatment/dose) will beadministered every 3 days beginning at the peak of EAE and continuedthrough the course of the experiment.

Assessment of Treatment Effects.

The inventor will evaluate EAE severity by monitoring clinical scores.The degree of inflammation and demyelination in brains and spinal cordsfrom these mice will be assessed by staining paraffin embedded sectionswith hematoxylin and eosin (to assess inflammatory infiltrates) andLuxol Fast Blue (to assess myelin integrity). The inventor will alsodetermine effects these treatments have on B-cell development andfunction. Specifically, the following will be assessed: (1) Changes inB-cell cytokines, BAFF and APRIL, by qPCR, ELISA and/or Western blot;(2) Changes in B-cell subsets infiltrating brain and spinal cords byflow cytometry; and (3) Changes in the development of anti-myelinautoantibodies in serum by ELISA.

Outcome Measures and Statistical Analysis.

For all EAE experiments, differences in clinical outcomes will bedetermined by Mann-Whitney test, with p<0.05 considered significant.ELISA, flow cytometry and qPCR results will be presented as means+/−onestandard deviation, and significance will be determined by a two-tailedStudent's t test or one-way ANOVA.

Power Calculations Determining Number of Mice Per Experiment.

Assuming that the variability in the proposed experiments will besimilar to that of the 5 most recent EAE experiments, 11 mice will berequired per experimental arm in order to detect a true difference of 1normalized unit at a significance level of 0.05 and power of 0.83 with astandard deviation of 0.3 units for each group of animals. The inventorintends to repeat each experiment two to three times.

Example 2—Results

The inventor's lab has developed and characterized two EAE models thatmimic different MS patient populations, TH1-induced EAE and TH17-inducedEAE. The inventor initially reported that TH1-EAE mimics an MS phenotypethat responds well to IFN-β treatment, whereas the TH17-EAE resemblespatients that are IFN-β non-responders (Axtell et al., 2010). Theinventor further characterized the phenotypic differences of these twomodels and found that mice with TH17-EAE have increased disease severity(FIG. 1A) and increased neutrophil infiltration in the CNS compared tomice with TH1-EAE (Herges et al., 2012). The inventor now has datademonstrating that B-cells have differing functions in these two EAEmodels. The inventor observed that the expression levels of BAFF andAPRIL RNA are significantly elevated in the spinal cords of mice withTH17-EAE compared to mice with TH1-EAE (FIG. 1B). In addition, theinventor found that the frequency of CD19⁺ B-cells were elevated inspinal cords of TH17-EAE compared to TH1-EAE, although frequencies ofCD4⁺ T-cells were equivalent in both EAE models (FIGS. 1C-D).

Since BAFF, APRIL and B-cells were elevated in the spinal cords of micewith TH17-EAE, the inventor assessed the effects recombinant TACI-Igtreatment had on this EAE model. The inventor treated TH17-EAE withTACI-Ig (100 μg/dose, N=3) or PBS (N=3) every 3 days beginning at thepeak of acute disease. The inventor found that mice treated with TACI-Ighad a greater recovery from paralysis compared to the PBS treated mice(FIG. 2A). The inventor also assessed the effects this treatment hadB-cell populations in the peripheral blood. The inventor found thatTACI-Ig treatment restored the frequency of immature B-cells(IgM^(high)IgD^(low)) in the blood to levels that are comparable tohealthy mice.

The inventors found that TH17-EAE represents a disease phenotype that issimilar to NMO where-as TH1-EAE represents MS. First, they found thatthe TH17 cytokines IL-17A and IL-17F are elevated in the serum from NMOpatients compared to MS patients (FIGS. 3A and B). Second, they foundthat like NMO, IFN-β treatment worsened disease TH17-EAE and conversely,like MS IFN-β reduced TH1-EAE (FIG. 4A-D). Third, like NMO, they foundthat TH17-EAE have more severe optic neuritis (FIG. 5A) and increasedvisual deficits (FIG. 5B) that TH1-EAE. Finally, they found that likeNMO, the neutrophils comprise a significant proportion of theinflammatory cells that infiltrate the central nervous system inTH17-EAE but not TH1-EAE (FIGS. 6 and 7B). In addition to neutrophils,they found that B-cells are a predominant infiltrate in CNS of TH17-EAEcompared to TH1-EAE (FIGS. 6 and 7C). Concordantly, they found that theexpression of BAFF and APRIL, which are cytokines that drive theproliferation and survival of B-cells, are significantly increased inTH17-EAE compared to TH1-EAE (FIG. 8). The inventors now find that thecell-type that expresses the majority of BAFF and APRIL in the lesionsof TH17-EAE are neutrophils (FIGS. 9A-B). These data demonstrates apathogenic cascade of events that occurs in TH17 mediatedneuro-inflammation such as NMO (FIG. 10). First, TH17 cells infiltratethe CNS and secrete TH17 cytokines (IL-17) which induces the expressionof neutrophil chemokines within the inflamed tissue. Neutrophils, thatare expressing high levels of BAFF and APRIL, then migrate to the CNSand induce the proliferation of B-cells in a BAFF dependent manner.These B-cells then can either present antigen to newly immigratedT-cells or express autoantibodies to promote more inflammation.

Example 3—Discussion

The data presented here support the hypothesis that B-cells, BAFF andAPRIL are inflammatory in TH17 mediated diseases and are converselyanti-inflammatory in TH1 mediated diseases. The expanded studies willdefinitively prove this hypothesis and in doing so it will provideinsights into (i) the inflammatory and regulatory functions B-cells havein autoimmune diseases; (ii) the role differential roles BAFF and APRILplay in TH1- and TH17-induced inflammation; and (iii) the clinicaleffects of blocking BAFF and APRIL in MS patients.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. Certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

V. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   U.S. Pat. No. 4,683,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,800,159-   U.S. Pat. No. 5,279,721-   Axtell et al., Nat Med. 16(4):406-12, 2010.-   Bellus, J. Macromol. Sci. Pure Appl. Chem., A31(1): 1355-1376, 1994.-   Herges et al., Mult Scler. 18(4):398-408, 2012.-   Innis et al., Proc. Natl. Acad. Sci. USA, 85(24):9436-9440, 1988.-   McDonald et al., Ann. Neurol., 50:121-127, 2001.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 1035-1038 and    1570-1580.-   Sambrook et al., In: Molecular Cloning, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N Y, 1989.

1. A method for treating a subject having neuromyelitis optica (NMO)comprising administering to said subject an inhibitor of B-cellactivating factor (BAFF) and/or an inhibitor or proliferating inducingligand (APRIL). 2-9. (canceled)
 10. The method of claim 1, furthercomprising administering to said subject a second NMO therapy. 11.(canceled)
 12. The method of claim 1, wherein said subject suffers fromvision impairment, muscle impairment or both.
 13. The method of claim12, wherein said subject, following treatment, exhibits an improvementin vision impairment, muscle impairment or both.
 14. The method of claim1, wherein said subject is a non-human mammal or a human.
 15. (canceled)16. A method for treating a subject having interferon-resistant multiplesclerosis (MS) comprising administering to said subject an inhibitor ofB-cell activating factor (BAFF) and/or an inhibitor or proliferatinginducing ligand (APRIL). 17-24. (canceled)
 25. The method of claim 16,further comprising administering to said subject a second MS therapy.26. (canceled)
 27. The method of claim 16, wherein said subject suffersfrom vision impairment, muscle impairment or both.
 28. The method ofclaim 27, wherein said subject, following treatment, exhibits animprovement in vision impairment, muscle impairment or both.
 29. Themethod of claim 16, wherein said subject is a non-human mammal or ahuman.
 30. (canceled)
 31. A method of identifying a subject havingmultiple sclerosis (MS) that will be resistant to interferon therapycomprising: (a) obtaining a sample from said subject; and (b) assessinglevels of a TH17/granulocyte factor, a type 1 interferon, B-cellactivating factor (BAFF) and/or proliferating inducing ligand (APRIL) insaid sample, wherein a subject having elevated TH17/granulocyte factors,type 1 interferons, BAFF and/or APRIL levels will not respond tointerferon therapy.
 32. The method of claim 31, further comprisingadministering to a subject having elevated levels an inhibitor of B-cellactivating factor (BAFF) and/or an inhibitor or proliferating inducingligand (APRIL).
 33. The method of claim 31, wherein assessing comprisesan immunoassay, mass spectrometry or RT-PCT. 34-35. (canceled)
 36. Themethod of claim 31, further comprising providing a written communicationof said level or levels. 37-44. (canceled)
 45. The method of claim 32,further comprising administering to said subject a second MS therapy.46. (canceled)
 47. The method of claim 32, wherein said subject suffersfrom vision impairment, muscle impairment or both.
 48. The method ofclaim 47, wherein said subject, following treatment, exhibits animprovement in vision impairment, muscle impairment or both.
 49. Themethod of claim 32, wherein said subject is a non-human mammal or ahuman. 50-55. (canceled)
 56. The method of claim 31, wherein more thanone of a TH17/granulocyte factor, a type 1 interferon, BAFF and APRILlevel is measured. 57-59. (canceled)
 60. The method of claim 31, whereinmore than one of a TH17/granulocyte factor, a type 1 interferon, BAFFand APRIL level are elevated.